The long-term objective of this project is to develop a novel intervention strategy to clear latent infections by herpes simplex virus (HSV). Latency is the most fascinating aspect of HSV biology and the most vexing aspect of HSV disease -- latency is why HSV infections have yet to be cured. The intervention strategy proposed here is: 1) Disrupt latency by antagonizing viral microRNAs (miRNAs) that we hypothesize repress lytic viral gene expression in latently infected neurons, using oligonucleotides that are complementary to these miRNAs (anti-miRs); 2) If necessary, further disrupt latency by antagonizing host functions that help maintain latency; and 3) Irreversibly inactivate the resultin replicating HSV using the anti-HSV drug acyclovir. The R21 phase of this project is a collaborative effort of three labs. The first aim is to determine whether selected anti-miRs or selected viral mutations affecting miRNAs or their target sites (Coen lab) derepress lytic gene expression in mouse and rat neuron in vitro models of HSV latency (Leib and Wilson labs). Gene expression will be measured using PCR-based methods (Coen and Wilson labs). Selected mutations will also be tested for their effects on lytic gene expression in an in vivo mouse latency model (Coen lab). The second aim is to test whether these anti-miRs, alone or in at least additive combination with small molecule antagonists of host functions, induce reactivation in the mouse and rat in vitro models, and whether acyclovir can cure latency in such models. The R33 phase of this project (Coen lab) focuses on efforts to cure latency in vivo in a mouse model. The third aim investigates anti-miRs from Aim 2, and comparable HSV mutations for effects on derepression of gene expression in this model. The fourth aim is to test whether these anti-miRs and comparable mutations, alone or in at least additive combination with small molecules from Aim 2 induce reactivation in the mouse model. The fifth aim is to test whether combinations identified in Aim 4 together with acyclovir can cure latency in vivo in the mouse model. These studies will be accompanied by pharmacokinetic assays of anti-miR and compound concentrations in plasma and tissues, and assays of toxicities. The goal is to achieve cure of HSV with minimal toxicity.